Acute hepatitis C virus (HCV) infection results in persistent infection in more than 60% of cases. In contrast, less than 30% of acutely infected patients resolve the infection spontaneously. We have previously shown that the cellular immune response may persist for more than 2 decades in spontaneously recovered patients and that it is impaired in patients with chronic hepatitis C virus infection. To analyze the very early factors that determine the outcome of infection we prospectively analyzed the humoral and cellular immune response in subjects with a documented exposure to HCV. HCV-specific cellular immune responses were detected within the first weeks after exposure even in the absence of humoral immune responses and targeted against structural as well as nonstructural HCV proteins with direct, ex vivo effector functions. The observation that HCV-specific T cell responses returned to baseline levels in all but 2 subjects within 12 weeks after exposure and that no subject developed persistent infection suggest that cellular immune responses contributed to early containment and rapid clearance of hepatitis C virus in these subjects and that humoral immune responses were not required. These findings led to the hypothesis that subjects who remain HCV-RNA negative and HCV antibody negative despite repeated exposure to HCV, are likely to maintain HCV-specific cellular immune responses. To investigate this hypothesis, we studied HCV-specific T cell responses of injection drug users who remained antiHCV negative, HCV RNA negative despite of having injected drugs (IDUs) for >15 years. This is an important study population, because less than 5% of IDUs remain HCV-seronegative after years of injection drug use. Our results show that that highly exposed, HCV seronegative IDUs display a cellular immune response in the blood that is quantitatively superior to that of persistently HCV-infected IDUs who are unable to clear the infection. The presence of cellular immune responses, especially those against nonstructural HCV sequences, indicates that exposure to HCV occurred but did not lead to persistent infection in this cohort of HCV seronegative subjects. Moreover, in related studies in chimpanzees, we demonstrated directly that the maintenance of multispecific and vigorous immune responses correlated with protection upon intravenous HCV rechallenge. Upon rechallenge of 3 HCV recovered chimpanzees, vigorous CD4+ T cell proliferation was followed by an increased frequency and a phenotypic and functional change of the tetramer+ CD8+ T cell population from CCR7-positive central memory to CCR7-negative, IFN-g-producing effector memory cells and rapid clearance of the rechallenge inoculum. Thus, the therapeutic induction of this type of memory responses and the generation of preventive vaccines and immunotherapies will be the main focus of future studies.